Morbidity and mortality from stroke and coronary artery disease (CAD) are probably the most critical public health problems in the US. Both ischemic stroke and coronary occlusion are complications of atherosclerosis and inflammation which culminate in platelet activation, recruitment and vascular occlusion - a breach of the control of blood fluidity. Advances in therapeutic modalities for stroke and CAD have reached a plateau with an absence of novel approaches. The broad, long term objectives and specific aims of this proposal are to bring the ecto-enzyme CD39/NTPDase1 to clinical fruition via the in vitro and ex vivo studies described herein. Our recent studies of CD39 have demonstrated that it constitutes the main control system for blood fluidity (thromboregulation). CD39 has a unique mode of action whereby it inhibits platelet activation and recruitment, acting in the fluid phase. This occurs via metabolic deletion of prothrombotic ADP, the final common mediator in platelet-induced vascular occlusion. Thus the mechanism of action of CD39 is radically different from currently available therapeutic modalities for treatment of stroke and CAD. Our successful treatment of stroke with the soluble form of CD39 (solCD39) in 3 animal models, as previously published, suggests its safety and efficacy. Extensive preliminary molecular biology data indicate that expression of different CD39 alternative splice variants regulates CD39 expression, assembly into cholesterol-rich domains, and enzymatic and biological activity. This enhances our comprehension of the mechanisms of action of the enzyme, and its regulation. CD39 will be studied in cryptogenic and atherothrombotic stroke patients. Thus, we will evaluate the profile of CD39 variant expression in normals and patients with cryptogenic and atherothrombotic stroke. Platelet reactivity and markers of platelet activation will be evaluated in our cryptogenic and atherothrombotic patients and controls using a spectrum of platelet agonists and coagulation parameters, including circulating tissue factor, in the setting of their medication status. This also includes characterization of leukocyte-platelet aggregates (with emphasis on monocyte-platelet aggregates) and vascular cell-derived microparticles. We anticipate that our research will afford novel therapeutic opportunities for CD39 in prophylaxis and management of stroke and CAD. In this application we have developed a critical mass of patients and investigators in order to further our comprehension of the pathogenesis and treatment of platelet-driven cardiovascular diseases and to develop CD39 as the prototype of the next generation of antithrombotic agents. Thus, this revised submission is a broadly based multidisciplinary, molecularly and clinically focused application. PUBLIC HEALTH RELEVANCE: Vascular occlusion in the brain (stroke) is initiated by excessive blood platelet reactivity and recruitment, leading to extensive morbidity and mortality. This reactivity can be neutralized by the enzyme CD39. The relevance of this research is that it is provides a new and safe approach to the management of stroke for which there is no satisfactory treatment. We anticipate that the research proposed herein will lead to formulation of a clinical trial of a human soluble apyrase such as solCD39.